In the fields of biochemistry, medical science, genomic drug discovery, etc., there is a strong demand for analyzing the structures of proteins, etc., in biological tissues and cells. One of the analyzing means for responding to this demand is mass spectrometry. In mass spectrometry, a test sample is irradiated with a laser to ionize biological molecules, etc., in the test sample, and the mass of the generated ions is analyzed (NPL 1).
Elucidating the localization of a target material in a biological tissue has great value in the fields of searching for abnormal materials, tracking pharmacokinetics, etc., in disease. Mass spectrometry is one of the methods of directly discovering and identifying a target material, and imaging mass spectrometry (IMS) has recently been suggested in which a target material in a biological tissue is identified in two dimensions, and the localization is elucidated (PTL 1 to 4, NPL 2 and 3, etc.).
When the distribution of a material in a biological tissue can be visualized and identified in two dimensions, information in vivo, e.g., identification of lesion sites and elucidation of disease-related materials (including intermediates), can be directly obtained, making significant contributions to society. Also, in the field of materials and nanotechnology, which has become increasingly sophisticated in recent years, analyzing the distribution and localization state of materials that help to increase functionality largely affects the expression of properties, production conditions, degradation state, etc. Thus, information obtained by IMS is extremely useful.
To perform mass spectrometry, a test sample needs to be ionized. Known methods of ionization include the use of ionization-assisting agent (matrix)-free secondary ion mass spectrometry (SIMS), and matrix assisted laser desorption/ionization (MALDI), which is capable of analyzing polymers by using a matrix.
These ionization methods are used in conventional IMS; however, the mass range that can be analyzed by SIMS is merely a mass-to-charge ratio (m/z) up to about 1,000. Moreover, most of the test sample is broken (by so-called fragmentation) in the ionization process, and when the test sample is a mixture, the spectra become complicated, making the analysis difficult. For this reason, the ionization method used in IMS is typically MALDI, in which test sample fragmentation rarely occurs; however, IMS using known MALDI has the following problem.
In the known MALDI, chemical synthesis materials (organic matrixes) and materials (inorganic matrixes) in which metal oxides or metal nanoparticles are dispersed in a solvent are known as an ionization-assisting matrix. Examples of organic matrixes include 1,8-dihydroxy-9(10H)-anthracenone (Dithranol), 2-(4-hydroxy phenylazo)benzoic acid (HABA), 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), etc., and the organic matrix is selected and used according to the analysis material (proteins, peptides, synthetic polymers, etc.).
In all conventional IMS, known organic matrixes for MALDI are used. However, since these organic matrixes were not originally developed for IMS, although the ionization efficiency is high, the matrix ability is reduced or lost in the presence of salt. Thus, these organic matrixes are not suitable for IMS that analyzes a crude test sample such as a salt-containing biological tissue. Further, since strong ion peaks originating from an organic matrix occur in a low molecular weight range (m/z: 700 or less), precise analysis is difficult when a target material is a drug, additive, etc., having a low molecular weight.
When a known organic matrix is used in INS, the matrix is added dropwise or injected in a liquid (solution) state to a test sample to incorporate an analysis subject. A crystal particle containing the analysis subject is then formed after drying. The crystal particle of the matrix obtained herein typically has a size of about 50 μm or more (NPL 4). Since the analysis subject is dispersed in the crystal particle of the matrix, even when the laser beam irradiation diameter for ionization is reduced, spatial resolution higher than the crystal particle size of the matrix cannot be obtained.
Further, when a liquid matrix is adhered to a test sample, the used liquid causes physical movement (so-called migration) of a target material, which causes the distribution information of the analysis subject to be lost. Moreover, the adhesion of a liquid matrix to a test sample allows crystal particles to cover tissue, which causes visual information to be lost, making identification of sites in the test sample difficult. In performing IMS, a test sample image is desirably observed by a CCD camera or microscope during analysis; however, it is difficult to identify which site is being imaged when the crystal particles of the matrix cover the test sample. It is also difficult to confirm after analysis from which site a target material is obtained.
On the other hand, even when an inorganic matrix obtained by dispersing metal nanoparticles in a solution is used, similar to the case where an organic matrix is used, migration of a target material occurs because of the use of a dispersion medium, such as hexane or alcohol, making it difficult to accurately analyze the localization.
As a liquid matrix-free technique, IMS using gold vapor deposition has recently been suggested (NPL 5). This method has a feature in that gold is vapor deposited on the surface of a test sample to assist ionization. This method, however, has room for improvement because special equipment is required for the vapor deposition of gold nanoparticles, and the peaks of the target material are reduced or made undetectable due to the strong ion peaks originating from the gold appearing in the spectrum.
Accordingly, development of an improved method for imaging mass spectrometry using an ionization-assisting matrix of a test sample is desired, wherein the ionization efficiency is high, migration and visual information reduction are inhibited, no interference peaks originating from the matrix occur, and the analysis can be performed at high spatial resolution.
Additionally, PTL 5 and NPL 6 disclose other conventional techniques involving the present invention. These documents disclose a laser desorption ionization (LDI) plate having platinum particles as ionization-assisting particles that assist the ionization of a test sample. However, although these documents disclose that a dispersion in which platinum particles are dispersed is used to support the platinum particles on a plate, they do not suggest physical vapor depositing platinum on the surface of a test sample or applying it to imaging mass spectrometry.